Papillomaviruses (PVs) infect the epithelia of animals and man, where they generally induce benign proliferation at the site of infection. However, there is a strong association between malignant progression of human genital, anal and oropharyngeal lesions and certain human papillomavirus (HPV) types, most frequently HPV 16. Our research is primarily concerned with development of vaccines and other infection inhibition strategies against HPV and the elucidation of the HPV life cycle. We have developed a simple and efficient strategy for generating high titers of infectious papillomavirus particles that transduce encapsidated marker plasmids, i.e. pseudovirions. We have exploited this technology in our basic virologic and translational research efforts. We have used our pseudovirus technology to develop the first cervicovaginal challenge model for HPVs. We have used this assay to define the molecular mechanism used by HPV to infect its target tissue and to determine how the antibodies induced L1- and L2-based prophylactic vaccines prevent infection. Key to the process is an obligatory binding to the basement membrane of a disrupted epithelium. This binding induces a conformational change required for subsequent steps in infection. The in vitro neutralizing assays we previously developed for L1 VLP vaccine analysis have proven to be relatively insensitive measures of protective L2 antibodies. Based on our understanding of the in vivo infectious process, we have recently developed a novel in vitro neutralizing assay that is 1000-fold more sensitive measure of L2 antibody activity. This assay will be critical in the further clinical development of L2-based vaccines, which we previously discovered to induce antibodies that, unlike L1 VLP vaccines, broadly cross-neutralize divergent mucosal and cutaneous HPV types. Our development of a method to induce efficient HPV pseudovirus infection of the female genital tract after transient disruption with the over-the-counter spermicide nonoxonol-9 has proven to be the key to our recent development of an effective, and we believe practical, intravaginal vaccination strategy. We have found that intravaginal pseudovirus vaccination of N-9 treated mice induces strong systemic and mucosal T and B cell responses to target antigens transduced by the pseudovirions. Systemic responses rival those induced by previously optimized Ad5 vectors. Intravaginal responses are remarkably strong, with up to 80% of all intravaginal CD8 T cells staining tetramer positive for the targeted antigen. Most of the induced T appear to be intraepithelial, and high level of effector memory CD8 T cells are maintained in the vaginal tract 100 days after vaccination. Critically, CD8 IEL's were not induced after systemic vaccination with Ad5 vectors. Intravaginal pseudovirus vaccination is a promising approach for focusing immune responses to the female genital tract and so might increase the effectiveness of vaccines directed against HSV and HIV infections and against HPV induced neoplasia. This concept was tested in an SIV/rhesus macaque intravaginal challenge model in collaboration with Dr. Franchini. In collaboration with Dr. Jeff Cohen, vectors expressing HSV antigens are being tested in mouse and guinea pig HSV-2 challenge models. We have determined that intravaginal delivery of Adenovirus 5 vectors induce similar locals T cell responses as intravaginal delivery of HPV pseudovirions and are superior at inducing systemic T cell responses. In collaborated with Crucell/Johnson & Johnson, we have determine that Ad26 and Ad35 vectors behave similarly. Crucell has extensive expertise in GMP production of their adenovirus vectors, which should greatly facilitate translation of our findings into a clinical trial. To more generally evaluate the potential of HPV pseudoviruses as gene transfer vehicles, we have conducted a broad infection tropism survey. In patent pending studies, we demonstrated that intact murine epithelium at all sites, whether simple, columnar, or squamous, was highly resistant to both virion binding and infection, whereas disrupted epithelium was susceptible. In contrast, virtually all human-derived epithelial cell lines in the NC1-60 panel were highly susceptible to infection in vitro. The remarkable specificity of HPV pseudovirus binding and infection appears to be mediated by specific heparan sulfate modifications of proteoglycan on the tumor cell surfaces that mimic those normally found on the basement membrane. The results suggest that HPV pseudovirions may be useful in tumor diagnostic or cytotoxic gene or drug therapy applications. In proof of concept studies, we documented highly specific binding and infection, and dramatic imaging, of human ovarian tumor nodules implanted in nude mouse peritoneum after intraperitoneal injection of RFP-expressing pseudovirus. In a preliminarly study using a mouse model of ovarian metastases, intraperitoneal injection of Herpes TK-expressing HPV psuedovirions followed by ganciclovir treatment, increased survival of tumor bearing mice. A manuscript reporting these findings is being prepared. A CRADA with Aura Biosciences was initiated to facilitate further development and clinical testing of this approach to tumor therapy. With CCR's Martin Brechbiel, we are investigating coupling radioactive alpha emitters for tumor killing. With CCR's Peter Choyke, we are investigating tumor imaging and killing by capsids coupled to an infrared dye, IR700. We have begun a virologic and immunologic characterization of a recently discovered papillomavirus for the domestic mouse. Large papillomas are induced on the muzzles and tails (but interesting not the backs) in immunodeficient strains but not in any of the many immunocompetent strains that we have tested. Both CD8 and CD4 T cells are involved in control of papillomavirus formation. We can now take advantage of the large set of immunological reagents and genetically modified mouse strains to provide the first in depth understanding of the immunological control of papillomavirus infection. A long standing collaboration with DCEG colleagues have resulted in several publications related to the NCI-sponsored prophylactic HPV vaccine clinical trial in Costa Rica. Highlights include demonstration of strong vaccine-type specific protection for four years in women receiving only one or two doses of the vaccine, remarkable persistence of the antibody responses after only one dose, and protection from oral infection by the vaccine targeted types.